High Resolution Spatial Transcriptomics using seq-FISH+

使用 seq-FISH 进行高分辨率空间转录组学

• 批准号: 10703380
• 负责人: John E Harris
• 金额: $ 36.4万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-12 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10703380
• 关键词: Address; Affect; Antibodies; Atlases; Autoimmunity; Biological Process; Biopsy; Bulla; Cell Communication; Cells; Center for Translational Science Activities; Clone Cells; Communication; Complement; Complex; Data; Dendritic Cells; Dermal; Epidermis; Equilibrium; Fluorescent in Situ Hybridization; Freezing; Gene Targeting; Genes; Genetic Transcription; Goals; Image; Immune; In Situ; Individual; Inflammation; Inflammatory; Knowledge; Label; Learning; Lesion; Ligands; Location; Maps; Messenger RNA; Microscope; Modification; Normal tissue morphology; Organ; Pathogenesis; Pigments; Population; Positioning Attribute; Privatization; Process; Punch Biopsy; RNA; Receptor Cell; Research; Resolution; Signal Transduction; Skin; Spatial Distribution; Structure; T cell clonality; T cell receptor repertoire sequencing; T-Cell Receptor; T-Lymphocyte; Techniques; Technology; Tissues; Transcript; Vitiligo; White Spots; cell type; cellular imaging; design; health disparity; immune cell infiltrate; improved; in silico; innovation; insight; interest; keratinocyte; melanocyte; multiple omics; receptor; receptor expression; single-cell RNA sequencing; skin disorder; transcriptome; transcriptomics; treatment strategy

Much has been learned about mechanisms that drive autoimmunity by studying vitiligo, a dis- ease of the skin that results from destruction of pigment-producing melanocytes. This results in disfiguring white spots that are particularly devastating for those with darker skin, which thus leads to health disparities for the most vulnerable of our population. We generated an extensive set of preliminary data using single cell RNA sequencing (scRNA-Seq) that revealed hun- dreds of conversations that are unique to vitiligo lesions involving every cell type of the epider- mis. These data indicate that cells and their signaling networks within affected and unaffected vitiligo skin are in fact more complex than previously appreciated. Yet because scRNA-Seq re- quires disruption of the tissue, the position of each cell and their communications within the skin remains unknown. We hypothesize that cells connected in silico by matching induced ligand-receptor expression are in close physical proximity in situ, and that T cell clones marked by identical T cell receptor (TCR) sequences associate in physical contact with melanocytes. The objective of the High- Resolution Spatial Transcriptomics Research Core using sequential fluorescence in situ hybridi- zation (seqFISH+) is to use our highly innovative platform to provide spatial context to T cell clonality, cellular positioning, and cell-cell communications revealed by the complementary, syn- ergistic Center Projects 1+2. Our seqFISH+ platform provides a cutting-edge, innovative ap- proach to map RNA expression in tissues with high accuracy and sub-diffraction-limit resolution, allowing for identification of cell classes, expressed genes, and spatial organization in tissues. This approach will be applied in the following specific aims: 1) Identify the position and distribu- tion of T cell clones marked by unique TCR sequences within tissue; and 2) Reveal the spatial organization of communicating cells by receptor-ligand mapping directly within vitiligo lesions. We will use punch biopsies taken from the same vitiligo lesions that have been analyzed by sin- gle cell techniques in each Project. We will design seqFISH+ probes that complement TCR se- quences and ligand-receptor pairs for hybridization, which will indicate the spatial distribution of T cell clonality and cell-cell communications through ligand-receptor pairing. This Research core combines a highly innovative conceptual approach with cutting-edge technology. The prelimi- nary data, investigative team, and innovative plan provides a robust and unique opportunity for discovery of the fundamental mechanisms by which immune cells target self-tissues, which may lead to improved treatment strategies.

关于通过研究白癜风来驱动自身免疫的机制，已经了解了很多 由于产生色素的黑素细胞破坏而导致的皮肤。这导致 毁容对皮肤较暗的人特别毁灭性的白点，因此 导致最脆弱的人群的健康差异。我们产生了广泛的 使用单细胞RNA测序（SCRNA-SEQ）的一组初步数据，该数据显示 白癜风病变独有的对话dreds，涉及Epider的每种细胞类型 误。这些数据表明，细胞及其信号网络在受影响和不受影响的情况下 实际上，白癜风皮肤比以前所欣赏的要复杂。然而，因为scrna-seq重新 组织中断，每个细胞的位置及其在皮肤中的通信 仍然未知。 我们假设通过匹配诱导的配体受体表达连接的细胞在计算机中连接 原位的物理接近近距离，而T细胞克隆以相同的T细胞受体为标志 （TCR）与黑素细胞物理接触的序列。高级的目标 分辨率空间转录组学研究核心使用顺序荧光原位杂交 Zation（Seqfish+）是使用我们高度创新的平台为T细胞提供空间上下文 互补，合成揭示的克隆性，细胞定位和细胞通信 精神中心项目1+2。我们的seqfish+平台提供了尖端，创新的ap- 以高精度和亚分形式限制分辨率绘制组织中的RNA表达， 允许在组织中鉴定细胞类别，表达基因和空间组织。 该方法将在以下特定目的中应用：1）确定位置和分布 T细胞克隆以组织内部独特的TCR序列标记的TION； 2）揭示空间 通过直接在白癜风病变中的受体映射来组织通信细胞。 我们将使用从相同的白癜风病变中进行的打孔活检。 每个项目中的GLE细胞技术。我们将设计与TCR Se-相辅相成的Seq鱼+探针 质量和配体受体对杂交，这将表明空间分布 通过配体配对T细胞克隆性和细胞电池通信。这个研究核心 将一种高度创新的概念方法与尖端技术结合在一起。前 NARY数据，调查团队和创新计划为可靠的独特机会 发现免疫细胞靶向自我影响的基本机制的发现，这可能 导致改进的治疗策略。